Immunosensors for Pesticide Analysis: Antibody Production and Sensor Development

ABSTRACT:?

Immunosensors, a type of affinity biosensor, are based on the binding interactions between an immobilized biomolecule (antibody/antigen) on the electronic transducer surface with the analyte of interest (antigen/antibody), resulting in a detectable signal. The sensor system takes advantage of the high selectivity provided by the molecular recognition characteristic of an antibody, which binds reversibly with a specific antigen.

This review article presents the current status of immunosensors, highlighting their potential benefits and limitations for pesticide analysis. The basic criteria for generating specific antibodies against low-molecular-mass pesticides, which are usually nonimmunogenic in nature, are briefly discussed. The article also describes the fundamentals of important transducer technologies and their use in immunosensor development.     

IgE and IgG are synergistic in antigen-mediated release of thromboxane from human lung macrophages.

The mechanism of IgE-mediated release of thromboxane A2 from human lung macrophages has been studied using a monoclonal chimeric human/mouse IgE antibody and its specific antigen. The cells could be sensitized at 37 degrees C but not at 4 degrees C by incubation with IgE, and released a significant amount of thromboxane A2 (TXA2), measured as the stable hydrolysis product TXB2, in response to an anti-chimeric IgE antibody. In contrast, stimulation of IgE-sensitized macrophages with the specific antigen produced less than 10% of this response. A similar time course for the release of TXB2 and the formation of inositol monophosphate in the presence of LiCl was observed. Cleavage of the Fc domain of the anti-chimeric IgE antibody substantially eliminated its capacity to stimulate IgE-sensitized cells. However, the weak or undetectable response to chimeric IgE plus specific antigen was substantially potentiated by an antigen-specific chimeric IgG antibody. IgG-sensitized macrophages did not respond to antigen challenge by the release of TXB2. Preincubation of the cells with a monoclonal antibody against the low affinity receptor for IgE (Fc epsilon RII/CD23) did not prevent IgE sensitization. We conclude that cell-bound IgE antibody cannot induce the release of TXB2 but has fixed antigen which then must interact with specific IgG antibody and IgG receptors to induce mediator release.     

The thermodynamics of hapten and antigen binding by rabbit anti-dinitrophenyl antibody.

Rabbit anti-dinitrophenyl antibody from a serum pool was obtained as five fractions of purified specific antibody by a limiting antigen precipitation method. Each fraction had a different binding affinity for epsilon-N-2,4-dinitrophenyl-L-lysine. The free energy changes for hapten binding to the five antibody fractions varied from -8.35 to -10.0 kcal/mol. An average deltaH of -13.9 kcal/mol was measured for the fractions with a batch calorimeter. The results indicate no significant correlation between enthalpy changes and free energy changes. However, a statistically significant correlation between the free energy changes and the entropy changes was found. The enthalpy of the anti-dinitrophenyl antibody interaction with multivalent dinitrophenyl human serum albumin was determined. These are the first enthalpy measurements of an antibody antigen reaction in which the intrinsic binding enthalpy between the antibody and the determinant group is known. The deltaH for the antigen binding reaction was -10.1 kcal/mol which is 3.8 kcal/mol less exothermic than the deltaH for the hapten binding reaction. The interactions that could lead to such a difference in enthalpy are discussed.     

Epitope mapping by surface plasmon resonance in the BIAcore

An epitope may be defined as a specific site on an antigen module characterized by the binding of one monoclonal antibody (MAb). Epitope mapping by surface plasmon resonance in the BIAcore biosensor may be performed to characterize an antigen or a group of specific MAbs or both. This article describes the BIAcore instrument and methods for such mapping. Examples include molecular interaction studies with simple and complex proteins, such as myoglobin and calprotectin, respectively.     

Selection of human antibody fragments by phage display

Here, we describe a protocol for the selection of human antibody fragments using repertoires displayed on filamentous bacteriophage. Antigen-specific clones are enriched by binding to immobilized antigen, followed by elution and repropagation of phage. After multiple rounds of binding selection, specific clones are identified by ELISA. This article provides an overview of phage display and antibody technology, as well as detailed protocols for the immobilization of antigen, the selection of repertoires on purified or complex antigens and the identification of binders.     

Modification of immune reactions by antigen-immunosuppressive agent conjugates. V. Studies on antigen-specific activity of 6-mercaptopurine bovine gamma globulin conjugates on the humoral immune response in rabbits]

Rabbits treated daily and for seven consecutive days with 6-mercaptopurine bovine gamma globulin conjugates (MPI--n-BGG; 'I'--characterizes the king of chemical binding and 'n' the number of coupled MP-residues per one mole BGG) show an altered immunological reactivity. A following intradermal immunization with BGG alum adsorbate results in a suppressed anti BGG antibody production on the third day after antigen application (antibody titer 1:320, antibody titer of control animals--pretreated with BGG and uncoupled 6-MP equals 1:5120). Already three days later the antibody titers of the test groups show a significant increase and are two dilution stages higher than the titers of the controls. A suppressive effect on the third day is induced by MPI--13-BGG, MPI--26-BGG, MPI--36-BGG and MPI--46-BGG; the later adjuvant effect can only be seen in the MPI--26-BGG, MPI--36-BGG and MPI--46-BGG but not in the MPI--13-BGG pretreated animal group. While the short time suppression was antigen specific--the humoral immune response against a second unrelated antigen was not reduced--the adjuvans effect was not antigen specific. A pretreatment with the substances mentioned above results in an increased anti BGG and anti HSA serum antibody level. Comparing investigations on the unspecific immunosuppression in rabbits by 6-mercaptopurine shows that application of 10 mg 6-MP/kg/day for seven days at first leads to a suppression but later on to an enhancement of antibody production. Application of 10 mg 6-MP/kg/day for 10 days results in a long lasting suppression without enhancement effect. As a reason for these differences the different catabolism of immunosuppressive agent and antigen is discussed. For the phenomena following antigen specific immunosuppression, similar mechanisms can be responsible.     

A common high molecular weight antigen of Babesia bovis isolates from Mexico

Cattle from an area of Mexico endemic with Babesia bovis infections have a dominant antibody response to a 152kDa antigen of the Tamaulipas strain of B. bovis. A mAb termed PB/5, showing a specific reactivity to this 152kDa antigen in Western blots, was identified. The mAb which reacted with the blunt end of B. bovis in an indirect fluorescent antibody test also reacted to a 152kDa antigen in two other isolates (Nuevo Leon and Yucatan), and a 175kDa antigen in the Huasteca B. bovis isolate from Mexico. Polyclonal monospecific sera from a calf inoculated with mAb-affinity purified 152kDa antigen (Tamaulipas strain) identified B. bovis by the indirect fluorescent antibody test and two antigens of B. bovis (65kDa and 152kDa) in Western blot. Since the epitope reacting to the mAb PB/5 is conserved, this antigen provides a basis for developing a diagnostic test or an immunogen.     

Autodisplay of 60-kDa Ro/SS-A antigen and development of a surface display enzyme-linked immunosorbent assay for systemic lupus erythematosus patient sera screening

Enzyme-linked immunosorbent assay (ELISA) is a common tool to test human sera on an antibody reaction against a specific antigen. The 60-kDa Ro/SS-A antigen for autoantibodies can be found in sera from systemic lupus erythematosus (SLE) patients. As in the case of 60-kDa Ro/SS-A, antigens used in ELISAs are recombinantly expressed in Escherichia coli and time-consuming purification steps are needed to get the proteins. To avoid these disadvantages, 60-kDa Ro/SS-A was expressed on the surface of E. coli using autodisplay, an efficient surface display system. Cells displaying 60-kDa Ro/SS-A on the surface were applied as an antigen source instead of the purified antigen. In total, 39 patients and 30 control sera were screened on a 60-kDa Ro/SS-A antibody reaction. To eliminate antibodies against native E. coli, human sera were preabsorbed with E. coli cells prior to the assay. The new ELISA protocol (surface display ELISA [SD-ELISA]) using E. coli with autodisplayed 60-kDa Ro/SS-A showed a sensitivity of 86.67% and a specificity of 83.33% by a cutoff value of 0.28. Our results show that autodisplay provides simple, rapid, and cheap access to human antigens for an ELISA to screen human sera against specific antibody reactions.     

The monoclonal antibody 22/18 recognizes a conformational change in an intermediate filament of the newt,Notophthalmus viridescens,during limb regeneration

Summary Previous work has shown that the monoclonal antibody 22/18 identifies progenitor cells (blastemal cells) which depend on the nerve for their division in the early stages of limb regeneration in the newt,Notophthalmus viridescens. This antibody also reacts with cultured cells derived from the newt limb, and the intensity of immunoreactivity appears related to cell density and differentiation into myotubes. We report here that the monoclonal antibody 22/18 recognizes a polypeptide (22/18 antigen) which is intracellular and filamentous. Double staining of cells with 22/18 monoclonal antibody and antibodies against various cytoskeletal components indicates that the epitope is expressed on an intermediate filament component. Although this antibody is specific for blastemal cells in cryostat sections of the regenerating limb, its reactivity on immunoblots is not confined to this tissue. The 22/18 antigen is differentially affected by aldehyde fixatives distinguished by the spacing of their reactive groups. While formaldehyde fixation impairs detection of the antigen, ethylene glycol-bis[succinic acid n-hydroxysuccinimide ester] reveals the antigen in sections of normal and regenerating limbs in a distribution that is consistent with the one obtained from immunoblots. We suggest that the 22/18 monoclonal antibody detects a change in protein conformation, probably related to changes in the physiological state of the cell, that occurs transiently during regeneration and possibly during development.     

An impedimetric immunosensor for the label-free detection of bisphenol A

Label-free detection of bisphenol A based on the impedance measurement was achieved with an impedimetric immunosensor. The immunosensor was fabricated by the covalent bond formation between a polyclonal antibody and a carboxylic acid group functionalized onto a nano-particle comprised conducting polymer. By using a commercial reagent 4,4-bis(4-hydroxyphenyl) valeric acid (BHPVA), which has an analogous structure of BPA, we have prepared the antigen through the conjugation of BHPVA with bovine serum albumin (BSA) and then produced a specific polyclonal antibody. The immobilization of antibody and the interaction between antibody and antigen were studied using quartz crystal microbalance (QCM) and electrochemical impedance spectroscopic (EIS) techniques. The impedance and mass changes due to the specific immuno-interaction at the sensor surface were utilized to detect antigen and bisphenol A (BPA). The immunosensor showed specific recognition of BPA with less interference than 4.5% from other common phenolic compounds. Under an optimized condition, the linear dynamic range of BPA detection was between 1 and 100 ng/ml. The detection limit of bisphenol A was determined to be 0.3+/-0.07 ng/ml. The proposed immunosensor was applied to a human serum sample and the BPA concentration was determined by the standard addition method.     

Effect of immunization with lipid associated polysaccharide antigen and anti CD-2 antibodies on class II MHC expression and cellular immune response in BALB/C mice infected with Leishmania donovani

In a bid to characterize the antigens and immunization mechanisms which may be used to produce a protective response against L. donovani, role of lipid associated polysaccharide (LPS) antigen and whole antigen was evaluated. BALB/C mice were immunized with whole or LPS antigen in combination with one of three putative adjuvents (anti CD-2 antibody/FIA/0.85% Saline). LPS antigen emulsified in anti CD-2 antibody was found to induce significant antibodies in mice on day 28 against challenge with lethal dose of L. donovani. Immunoprophylactic properties of LPS and whole antigen was investigated on day 40 through cytokine elicitation (IL-2), MIF) in culture supernatants of spleen cells, but before that MHC-II expressed on macrophage was studied. The LPS antigen in combination with anti CD-2 antibody was found to be most immuno-reactive inducing higher MHC-II expression on macrophages which was associated with substantial rise in the level of MIF and IL-2. It coincided with decline in antibody titre in 100% mice immunized with LPS antigen while Leishmania injected as whole antigen failed to induce specific macrophage and T-cell response with all the above formulations. We surmise from our data that lipid associated polysaccharide antigen linked to anti CD-2 antibody has potential for eliciting protective immunity against Leishmania.     

Kinetics of the antibody response to type III pneumococcal polysaccharide (SSS-III). I. Use of 125I-labeled SSS-III to study serum antibody levels, as well as the distribution and excretion of antigen after immunization.

A simple method was described for the preparation of 125I-labeled type III neumococcal polysaccharide (SSS-III) with a high specific radioactivity which retained the physical and immunologic properties of native SSS-III. SSS-III was used to study the serum and tissue levels of antigen, as well as its excretion, after i.p. injection. When an optimally immunogenic dose (0.5 mug) of antigen was given, greater than 90% of the injected antigen was excreted during the first 3 days after injection; however, after day 3, the SSS-III which remained in each mouse was firmly bound to various tissues, and less than 5 ng SSS-III was released into the circulation daily. SSS-III was also used in a Farr test to measure serum antibody levels; the kinetics for the appearance of PFC/spleen and serum antibody levels were measured at 24-hr intervals after immunization with 0.5 mug of antigen. Maximum PFC/spleen were observed on day 4 after immunization whereas the peak serum antibody level was seen on day 5. The decay of serum antibody level from its maximum value was much slower than that of the PFC/spleen. The data describing the distribution of SSS-III in vivo and the measurement of serum antibody levels indicated that treadmill neutralization was not a factor in determining the serum antibody levels after immunization with an optimally immunogenic dose of SSS-III.     

A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture.

Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18 IU. The results indicated a good correlation with the NIH mouse potency assay (r=0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation.     

A rapid method to improve protein detection by indirect ELISA

The enzyme-linked immunosorbant assay (ELISA) is a rapid, high-throughput, quantitative immunoassay for the selective detection of target antigens. The general principle behind an ELISA is antibody mediated capture and detection of an antigen with a measurable substrate. Numerous incarnations of the ELISA have resulted in its commercialization for sensitive diagnostic applications using a variety of detection platforms. Many of these applications require a pair of antibodies necessary for the capture and detection of a specific antigen (cELISA) in defined substrates. However, the availability of cELISA for target antigens is limited and thus restricts the use of this technique for quantitative measure of antigens during discovery. Alternatively, the indirect ELISA (iELISA) requires only a single antibody directed against a target antigen that has been immobilized to a surface. Unlike the cELISA, which uses an immobilized capture antibody that can bind a native antigen in solution followed by a detector antibody that binds captured antigen, the iELISA uses an antibody the binds directly to an immobilized antigen for detection. Although the iELISA may lack the sensitivity of a cELISA, its requirement of only a single antigen specific antibody makes it a simple technique for evaluating the relative difference in the level of target protein expression between samples. However, many antibodies that work effectively to detect protein antigens in other immunoassays such as Western blotting or immunohistochemistry fail to work in microplate based iELISA. Although these alternate immunoassay methods are useful for qualitative determination of target antigens, they provide limited quantitative information, limiting the assessment of sample specific differences in protein expression. We hypothesized that protein conformation following adsorption on the plastic surface of microplates impedes antibody epitope binding and this restriction could be overcome by a short chemical denaturation step. In this report we define a rapid method to assess the utility of an antibody for iELISA application and demonstrate a significant improvement in both qualitative and quantitative protein detection after chemical denaturation using defined assay conditions.     

Enzyme-linked immunoassay (ELISA) for connective tissue components

Enzyme-linked immunoadsorbant assays have been developed for types I, II, III, and IV collagen and for laminin and fibronectin. These assays offer a specific, sensitive, and convenient method for the measurement of various connective tissue components either separately or simultaneously. Plastic microtiter wells are coated with purified antigen, then antibodies to the antigen are allowed to bind to the insolubilized antigen in each well. The amount of bound antibody is determined by incubation with a second antibody which is covalently linked to alkaline phosphatase or horseradish peroxidase. The amount of bound enzyme is assayed after adding an appropriate substrate. The level of protein in a sample is estimated from its ability to inhibit the binding of the first antibody to the antigen-coated well. Specificity of the assay depends on the purity of the adsorbed antigen and allows for highly specific tests. Under optimum conditions the sensitivity of the assay is determined by the K_B of the antibody and allows 10–20 ng of a specific type of collagen or laminin and 1 ng fibronectin to be detected.     

鼠疫菌F1抗体/抗原酶联免疫诊断试剂盒的应用评价

目的对兰州生物制品研究所有限责任公司研制的鼠疫菌F1抗体酶联免疫诊断试剂盒和鼠疫菌F1抗原酶联免疫诊断试剂盒进行临床应用评价。方法采用双抗原/抗体夹心酶联免疫吸附试验(ELISA)、间接血球凝集试验(IHA)、胶体金免疫层析试验(GICA)3种方法的诊断试剂对比检测云南省地方病防治所中心实验室保藏的和现场采集的血清样品和脏器样品,对血清样品做鼠疫菌F1抗体检测,对脏器样品做鼠疫菌F1抗原检测。结果在358份血清样品中,ELISA试剂检出F1抗体阳性52份(14.52%),IHA试剂检出阳性37份(10.34%),GICA试剂检出阳性45份(12.57%)。ELISA与IHA试剂的符合率为95.23%,与GICA试剂的符合率为96.92%。经统计学χ2检验,ELISA试剂检出F1抗体阳性率高于IHA试剂(χ2=11.53,P=0.000 7),与GICA试剂检出的差异无统计学意义(χ2=3.27,P=0.070 4)。进一步分析滴度差值频数,ELISA试剂检测人血清的敏感性高于IHA试剂的样品占87.5%。在117份脏器样品中,3种试剂均检出F1抗原阳性15份(12.82%),符合率100%。滴度差值频数比较,ELISA试剂检测敏感性高于反向间接血球凝集试验(RIHA)试剂的样品为78.57%。结论兰州生物制品研究所有限责任公司研制的鼠疫菌F1抗体酶联免疫诊断试剂盒和鼠疫菌F1抗原酶联免疫诊断试剂盒性质特异,其敏感性优于IHA试剂盒和GICA试剂条,值得在鼠疫的监测和快速诊断中推广应用。     

Quantitative immunochromatographic analysis: theory and application to theophylline immunoassay

The development of an immunochromatographic technique suitable for rapid analysis of biological fluids is described. Quasi-one-dimensional antibody lattices specific for theophylline were constructed by packing Sepharose beads conjugated with specific antibody into specially designed narrow capillary tubes. The design of these capillary columns was such that they would subtract a preset threshold quantity of antigen (label and analyte) from the total amount presented. Labeled antigen, which appeared in the flowthrough, could then be used to precisely quantitate the analyte present. The ideal format would permit very precise subtraction of 100% of the available antigen up to the threshold amount and none of the remainder. The microcolumn described here comes close to this ideal behavior through the attainment of very high ratios of bound/free antigen. The elevated bound/free ratio could be explained by theoretical analysis of the effect on equilibria of the high antibody concentration in this quasi-one-dimensional system. Lattices containing anti-theophylline antibodies were used to develop a competitive enzyme immunoassay for theophylline which demonstrated a dose-response that was closely similar to that predicted by theoretical treatment. The entire assay procedure was performed in less than 30 min and demonstrated a sensitivity limit of approximately 20 ng/ml. Preliminary studies on clinical serum samples suggest that this assay has potential for the routine analysis of biological fluids.     

Growth promotion of genetically modified hematopoietic progenitors using an antibody/c-Mpl chimera

Thrombopoietin is a potent cytokine that exerts proliferation of hematopoietic stem cells (HSCs) through its cognate receptor, c-Mpl. Therefore, mimicry of c-Mpl signaling by a receptor recognizing an artificial ligand would be attractive to attain specific expansion of genetically modified HSCs. Here we propose a system enabling selective expansion of genetically modified cells using an antibody/receptor chimera that can be activated by a specific antigen. We constructed an antibody/c-Mpl chimera, in which single-chain Fv (ScFv) of an anti-fluorescein antibody was tethered to the extracellular D2 domain of the erythropoietin receptor and transmembrane/cytoplasmic domains of c-Mpl. When the chimera was expressed in interleukin (IL)-3-dependent pro-B cell line Ba/F3, genetically modified cells were selectively expanded in the presence of fluorescein-conjugated BSA (BSA-FL) as a specific antigen. Furthermore, highly purified mouse HSCs transduced with the retrovirus carrying antibody/c-Mpl chimera gene proliferated in vitro in response to BSA-FL, and the cells retained in vivo long-term repopulating abilities. These results demonstrate that the antibody/c-Mpl chimera is capable of signal transduction that mimics wild-type c-Mpl signaling.     

Immunoregulation by antigen/antibody complexes. I. Specific immunosuppression induced in vivo with immune complexes formed in antibody excess

Specific immune complexes, prepared at different ratios of antibody to antigen, were examined for their effects on the antibody response of BALB/c mice to the cell wall polysaccharide antigen (PnC) extracted from Streptococcus pneumonia R36a. Mice immunized with complexes formed in antigen excess developed a PnC-specific antibody response that was equivalent to that in mice injected with free antigen. On the other hand, mice injected with complexes formed in antibody excess developed very little PnC-specific antibody. Furthermore, administration of immune complexes (formed in antibody excess) resulted in suppression of the response to an immunogenic dose of PnC given concurrently or 1 day after injection of immune complexes but not when the antigen was given 1 day before injection of the immune complexes. Injections of free antibody (TEPC-15) also resulted in suppression of the response to antigenic challenge; however, suppression was greatest when the antibody was injected concurrently with the antigen, suggesting that the suppression was mediated through the formation of immune complexes in vivo. The suppression appears to be specific for the antigen (PnC), since in mice injected with TEPC-15/PnC complexes (formed in antibody excess) and challenged with PnC coupled to sheep RBC, only the response to PnC was suppressed.     

Epstein-Barr virus LMP2A enhances B-cell responses in vivo and in vitro

Epstein-Barr virus (EBV) establishes latent infections in a significant percentage of the population. Latent membrane protein 2A (LMP2A) is an EBV protein expressed during latency that inhibits B-cell receptor signaling in lymphoblastoid cell lines. In the present study, we have utilized a transgenic mouse system in which LMP2A is expressed in B cells that are specific for hen egg lysozyme (E/HEL-Tg). To determine if LMP2A allows B cells to respond to antigen, E/HEL-Tg mice were immunized with hen egg lysozyme. E/HEL-Tg mice produced antibody in response to antigen, indicating that LMP2A allows B cells to respond to antigen. In addition, E/HEL-Tg mice produced more antibody and an increased percentage of plasma cells after immunization compared to HEL-Tg littermates, suggesting that LMP2A increased the antibody response in vivo. Finally, in vitro studies determined that LMP2A acts directly on the B cell to increase antibody production by augmenting the expansion and survival of the activated B cells, as well as increasing the percentage of plasma cells generated. Taken together, these data suggest that LMP2A enhances, not diminishes, B-cell-specific antibody responses in vivo and in vitro in the E/HEL-Tg system.